Enzymes Activation and Deactivation

1. Enzymes Activation and Deactivation November 19th, 2012

2. Enzymes are Not Consumed in Reaction • https://www.youtube.com/watch?v=0XjyAkeQJag&feature=related

3. Factors Effecting Enzymes • Enzymes are not perfect • They respond to environmental conditions • They work depending on various factors which are? • ________, ___________, _____________ • Changing these factors effects? _____________ Enzyme under Stress

4. pH • An decrease in pH, increases the [H+] ion concentration in solution • An increase in pH, increases the [OH-] ion concentration in solution • These ions interfere with hydrogen bonds and ionic bonds • Changing the conformation of the enzymes specifically the active site • Activity of enzyme is affected

5. Optimal pH • Different enzymeshave different optimum pHs • At optimum pH the active site is the shapemost complementary to the shape of their Substrate • At optimum pH, the rate of reaction is highest • Large changes in pH can cause enzymes to Denature and permanently loose their function

6. Temperature Effects Enzymes • Generally, enzymes have a narrow range of temperature they work in, Why? • At the optimal temperature enzymes are most active

7. Increase Temperature • What increases?energy • Makes the substrate more active in solution. So? • More chances of substrate colliding with active site. • Makes the enzyme more flexible. • Puts strain on weaker bonds. • Pass a certain point enzymes denature. What does it mean to denature? active site changes

8. Decrease Temperature • Makes the enzyme less flexible, • Pass a certain point enzymes do not function properly • Not enough energy present

9. Taq Polymerase • Enzyme comes from ThermusAquaticus, a species that thrive in hot springs and heat vents. • Functions at high temperature • Used in Polymerase Chain Reaction (PCR) • Can make multiple copies of a DNA sample using only a small amount. • PCR can be used for a forensic investigation, genetic diseases, drug discovery and detection of pathogens

10. Temperature Curve • Various thermophillic organisms have their own type of DNA polymerase such as Pfu Polymerase (Pyrococcusfuriosus) versus Taq Polymerase

11. Regulation of Temperature • Endotherms can maintain body temperature • Heat is produced and regulated by the body, How? • Ectothermsdo not maintain a body temperature • Less sensitive to changes in body temperature • Endothermic organisms are mammals, birds and some fish • Most enzymes in the human body have an optimal temperature of 37°C

12. Concentration • What is concentration? • Will the concentration of substrate the rate of reaction? • Will the concentration of enzyme effect the rate of reaction?

13. Substrate Concentration • Adding more substrate increases rate of the reaction • More substrate molecule collide with active site • At a certain point adding more substrate has no more effect. Enzyme active site is saturated

14. Enzyme Concentration • If an enzyme is saturated what can you do? • Increasing enzyme concentration, increases the rate of reaction • Why does the graph level off?

15. Enzyme Regulation November 20th & 21st,2012

16. Biochemical Process • http://www.iubmb-nicholson.org/animaps.html

17. Enzyme Regulation • There are enzymes for each specific reaction of the human body • There is a need to control enzyme activity • Regulation is efficiency • Enzymes can be activated and inhibited Road Map

18. Aspirin • Cyclooxygenase 2 (COX2) makes prostaglandins • These chemical are involved in inflammation • Inflammation is felt as pain and swelling in body • Aspirin reacts with the amino acid serine irreversibly, blocking the active site, substrate can not bind • Other pain killers such as ibuprofen (Advil) bind less strongly, are reversible bound

19. Inhibition • Enzyme inhibitors are substances that interfere with catalysis • Inhibitors slow down the rate of reaction • Inhibitors can be reversible or irreversible • Irreversible inhibition – halts enzymatic reaction permanently • Reversible inhibition – slows down the reaction temporarily • Inhibitors can act in a competitive or non competitive form and interfere with the reaction

20. Competitive Inhibition • Competitive inhibition: Enzyme inhibitors prevent the formation of Enzyme-Substrate complexes because they have a similar shape to the substrate molecule. • Prevents enzyme from carrying out reaction it is suited for Inhibitor Substrate Inhibitor Competes with Substrate for the Active Site Enzyme with Active Site Specific for Substrate

21. Competitive Inhibition • Inhibitor has a different shape than the substrate but complements the active site • Inhibitor does not react since it has different structure than the substrate. • Reaction rate is decreased since fewer substrate molecules can bind to the enzyme • Inhibition is typically temporary, the inhibitor eventually leaves the active site • Inhibition depends on the relative concentrations of substrate and inhibitor, both compete for place in enzyme active site

22. Methanol Poisoning • Methanol if ingested is oxidized to formaldehyde and formic acid • Attack on the optic nerve causes blindness. • Methanol found in engine fuel, solvents, window cleaner, and antifreeze Source: http://curriculum.toxicology.wikispaces.net/2.2.5.2.5+Methanol

23. Ethanol Competes with Methanol • Ethanol competitively inhibits the oxidation of methanol by Alcohol Dehydrogenase • Ethanol is oxidized in preference to methanol • Oxidation of methanol is slowed down • Toxic by-products do not have chance to accumulate. Source: http://curriculum.toxicology.wikispaces.net/2.2.5.2.5+Methanol

24. E.Coli cells can not grow and die Pennicillin • Pennicillin, an antibiotic, works against disease causing bacteria • Stops cell wall cross-linking permanently • Inactivates transpeptidase, used to build cross-linked peptidoglycan layer in the membrane • The cross-linking peptide chains have repeats of D-Alanine • Pennicillin also has a repeat of D-Alanine-D-Alanine

25. SuccinateDehydrogenase Inhibitor • SuccinateDehydrogenase catalyzes the conversion of succinate to fumerate, an important biochemical reaction in cellular respiration. • Malonate inhibits this reaction competitively • Used to find active site chemistry • Used to study inborn errors of metabolism

26. Non-Competitive Inhibition • Non-competitive inhibition: enzyme inhibitors prevent the formation of Enzyme-Product Complexes. • Inhibitors prevent the substrate to react and form into product • Non-competitive inhibitors bind to a site other than the Active Site • Binding causes conformational changes that change the tertiary structure of the enzyme • Thus, enzyme can not catalyze reaction

27. Non-Competitive Inhibition Enzyme Active Site Complementary to Substrate Substrate Non-competitive Inhibitor

28. Non-Competitive Reaction • Since they do not compete with substrate molecules, non-competitive inhibitors are not affected by substrate concentration. • Many non-competitive inhibitors are irreversible and permanent, and effectively denature the enzymes which they inhibit. • However, there are a lot of non-permanent and reversible non-competitive inhibitors that are vital in controlling metabolic functions in organisms.

29. Cyanide Poisoning • Another enzyme found in cellular respiration is cytochromeoxidase, one of the most important enzymes in the electron transport chain of reactions that occurs in the mitochondria inner membrane • Here oxygen is reduced and 34 ATP molecules are made.

30. Cyanide Poisoning • Cyanide acts as a non-competitive inhibitor for cytochromeoxidase complex • Cyanide does not compete for the active sites of the enzyme because it has no similarity to the substrate cytochrome • Cyanide attaches to another site on the enzyme and disrupts the enzyme's shape. • This brings the electron transport chain to a halt • No energy can be derived out of respiration • Hydrogen cyanide inhibits metal-containing enzymes in the body, such as cytochrome c-oxidase, which contains iron

31. Irons in Enzymes • Chemical catalyst are usually metals • Many enzymes get their ability to catalyze reactions due to metals found in the active site • One common metal used is iron Fe2+ that is found in a protoporphyrin ring

32. Ferrochelatase • Ferrochetalaseinserts iron into protoporphyrin rings • Lead forms covalent bonds with the sulphydryl side chains of the amino acid cysteine in the enzyme and prevents catalytic activity • The binding of the heavy metal shows non-competitive inhibition because the substrate still has access.

33. Chymotrypsin • Chymotrypsin is an enzyme which hydrolyzes peptides bonds • In its active site there are three amino acids Histidine57, Serine195 and Asparagine102 known.

34. Hydrogen Ion Inhibits Chymotrypsin • These amino acids allow for the substrate to be cleaved. • By lowering pH, amino acids in the active site no longer accept hydrogen proton since Asp102 becomes protonated (hydrogens added) • Hydrogen ion acts as a non-competitive inhibitor by preventing catalysis but do not prevent the substrate from binding to the active site.

35. Biochemical Pathway • A biochemical pathway is a series of step reactions leading to a product • Enzymes lie in biochemical pathways • There are specific enzymes for each reaction step • Metabolism is a sum of biochemical pathways and is made of anabolic and catabolic processes

37. Need to Regulate • There are so many pathways that are incorporated in the metabolic system of the human body • An efficient process is needed to regulate the use of resources and ensure that only what is required is being produced or broken down • Enzymes can be regulated by the ability to be activated and deactivated when needed

38. Allosteric Enzyme Regulation • An allosteric site, a site away from the active site, can bind molecules to change conformation of the enzyme. • At the allosteric site for an inhibitor, binding of an inhibitor causes a conformational change such that the active sites of an enzyme are non complementary to the substrate. • An activator can bind to its allosteric site to open or improve the fit between substrate and enzyme.

40. Feedback Inhibition • In a biochemical pathway, by controlling an earlier step, the next series of reaction steps can be controlled • Usually the end product in a chain of reactions is an inhibitor of an earlier enzyme in the chain to stop the creation of more product • Process is self-regulating and cell resources are not wasted by making more product than needed

41. Feedback Inhibition Using an Allosteric Site

42. Regulation of Glycolysis • Glycolysis is biochemical process where glucose is broken down to pyruvate • Pyruvate is used in mitochondria in the process of aerobic respiration to derive ATP • Pyruvatekinase is the enzyme that converts phosphenolpyruvate to pyruvate in glycolysis • This enzyme is the third regulated enzyme of glycolysis • ATP and alanine act as allosteric inhibitors of pyruvatekinase

44. Feedback Inhibition of Pyruvate